Production Process Of Bio Fertilizers

1. Production Process Of Bio Fertilizers Name: Anirudh Mondal Course: B.Tech Biotechnology(2011-2015) University:Thapar University Patiala Punjab Roll No.: 701100004 Summer Internship Undertaken at RASHTRIYA CHEMICALS AND FERTILIZERS LTD. MUMBAI ( A Govt. Of India Undertaking ) UNDER THE SUPERVISION OF: Dr. Archana Kale Deputy Manager, R&D Division, RCF Trombay Unit apkale@rcfltd.com

2. CERTIFICATE This is to certify that Mr. Anirudh Mondal of ‘Thapar University, Patiala, Punjab’ ; pursuing Bachelors of Technology has satisfactorily completed his project on “PRODUCTION PROCESS OF BIO FERTILIZERS” under our supervision during the period from 3rd June to 3rd July at Rashtriya Chemicals and fertilizers Ltd Trombay Unit, Chembur , Mumbai – 400 074. Guide Dr. Mrs. Archana Kale Deputy Manager (R & D), Rashtriya Chemicals and Fertilizers Ltd. Trombay Unit

3. ACKNOWLEDGEMENT I would like to extend my appreciation and gratitude to Dr. Archana Kale (Dy. Manager R & D) for her assistance, able guidance, encouragement ,whole hearted cooperation and constructive criticism during all the paces of my project. I deeply express my sincere thanks to her for encouraging and allowing me to do the project in a real and live industrial environment on the topic “PRODUCTION PROCESS OF BIO FERTILIZERS” at the BIOLA Plant at RCF Trombay. I would like to express my gratitude to Mrs. Jagruti, R & D Department for her constant support throughout the project. I would also like to thank all the employees and the support staff at Rashtriya Chemicals and Fertilizers Ltd. for their motivation and support. Dr. Archana Kale (Deputy Manager, R&D Division, Rashtriya Chemicals and Fertilizers Limited)

4. TABLE OF CONTENTS

5. INTRODUCTION TO RASHTRIYA CHEMICALS AND FERTILIZERS LIMITED Rashtriya chemicals and fertilizers limited (RCFL) , is one of the leading public sector unit in India, directly under the ministry of chemicals and fertilizers, government of India based in Mumbai. RCFL is manufacturer of industrial chemicals and fertilizers with a “MINIRATNA" status awarded by the Government of India. RCFL was incorporated under the Companies Act 1956. RCFL currently has 2 Units: 1. Trombay Unit situated in Trombay (Chembur), Maharashtra. 2. Thal Unit situated at Alibaug, Rajgad, Maharashtra. Both the Units of RCFL are accredited with ISO-14001 certification for the environmental systems while Thal Industrial Products are accredited with ISO 9002 certification for quality control. Thal and Trombay Units have also received OHSAS-18001 certification. RCFL has constantly striven to maintain the environment in its vicinity. Several Projects notable among them being the Chembur Green Project at Trombay and the afforestation of Thal have been highly successful. RCFL has invested substantially in Pollution abatement schemes at Trombay and Thal through technology up gradation. RCF has won several awards for pollution control and clean technology. Notable awards being the Indira Gandhi Memorial award for Pollution control and the Rajiv Gandhi Award for clean technology awarded to Thal. RCFL has been an MOU signing Company since 1988-89. The Company also received MERIT CERTIFICATE for excellence in the Achievement of MOU targets for the year 2009-2010, 2010-11, 2011-12 and 2012-13.

6. Problem with Chemical Fertilizers • After the introduction of chemical fertilizers in the last century, farmers were happy of getting increased yield in agriculture in the beginning. • But slowly chemical fertilizers started displaying their ill-effects such as leaching out, and polluting water basins, destroying micro-organisms and friendly insects, making the crop more susceptible to the attack of diseases, reducing the soil fertility and thus causing irreparable damage to the overall system.

7. BIO FERTILIZERS- INTRODUCTION AND ADVANTAGES • A bio fertilizer is a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant • Bio fertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances • Bio fertilizers can be expected to reduce the use of chemical fertilizers and pesticides

8. Through the use of bio fertilizers, healthy plants can be grown, while enhancing the sustainability and the health of the soil • Since they play several roles, a preferred scientific term for such beneficial bacteria is "plant-growth promoting rhizobacteria" (PGPR) • They are extremely advantageous in enriching soil fertility and fulfilling plant nutrient requirements by supplying the organic nutrients through microorganism and their byproducts. • Bio fertilizers do not contain any chemicals which are harmful to the living soil. • Bio fertilizers eco friendly organic agro-input and more cost-effective than chemical fertilizers.

9. Since a bio fertilizer is technically living, it can symbiotically associate with plant roots. Involved microorganisms could readily and safely convert complex organic material in simple compounds, so that plants are easily taken up. Microorganism function is in long duration, causing improvement of the soil fertility • It maintains the natural habitat of the soil. It increases crop yield by 20-30%, replaces chemical nitrogen and phosphorus by 25%, and stimulates plant growth. It can also provide protection against drought and some soil-borne diseases. • Bio fertilizers are cost-effective relative to chemical fertilizers. They have lower manufacturing costs, especially regarding nitrogen and phosphorus use.

10. Manufacturers Of Bio-Fertilizers in India • Rashtriya Chemicals and Fertilizers Limited Mumbai • Agri Life Hyderabad • Mani Dharma Biotech Limited Chennai • Maharashtra Chemicals and Fertilizers Pune • Star Trace Private Limited Chennai • East Coast Seaweed Inc. Madurai • Shree Bio care India Ahmedabad

11. Production Scenario

12. Important Bio fertilizer Producing States

13. TYPES OF BIO FERTILIZERS • N2 fixing Bio Fertilizers. • P Solubilizing Bio Fertilizers • P Mobilizing Bio Fertilizers • Silicate Solubilizing Bio Fertilizers • Zinc Solubilizing Bio Fertilizers • Plant Growth Promoting Rhizobacteria • K solubilizing Bio Fertilizers • VAM Bio Fertilizers

15. Nitrogen Solubilizing Bio Fertilizers Rhizobium is a nitrogen fixing bio fertilizer. Rhizobium sp. is the symbiotic nitrogen fixer which assimilates atmospheric nitrogen and fixes in the root nodule, formed in the roots of leguminous plants. These bacteria infect the roots of leguminous plants, leading to the formation of “lumps” or “nodules” where the nitrogen fixation takes place. The bacterium also produces enzymes (nitrogenase) that supply a constant source of reduced nitrogen to the host plant.  Bio promoter-rhizobium is available as carrier based powder formulation and also in liquid formulation. Suitable for:Ground nut, Black gram, Green gram, Red gram, Cow pea, Bengal gram, Mustard, Soy bean, French bean, Cluster bean, Lab-lab, Sesbaniasp, Leguminous trees etc.

16. Azospirillum is a nitrogen fixing bio fertilizer. Nitrogen is a major nutrient for all plants. Azospirillum lipofereum is a very useful soil and root bacterium. It is an associative symbiotic nitrogen fixing bacteria. It is found in the soil around plant roots and root surface. It also produces growth-promoting substances like indole acetic acid (iaa), gibberellins, pantothenic acid, thiamine and niacin and it promotes root proliferation and it improve the plant growth yield. It increases the rootlet density and root branching resulting in the increased uptake of mineral and water. Suitable for: Millets, oilseeds, fruits and vegetables, sugarcane, banana, coconut, oil palm, cotton, chilly, lime, coffee and tea, areca nut and rubber, flower, spices, and condiments, herbs, lawns and ornaments, trees etc. 

17. N2 fixing Bio Fertilizers

18. Potassium Mobilizing Bio Fertilizer Potash Mobilizer is a beneficial bacterium capable of mobilizing Potassium available in soil into the root zone of plants. It works well in all types of soil. Use of such bacteria in powder form can increase the availability of more potash in usable form to the plants. Benefits:Mobilizes the insoluble potash in the soil into easily available form to the plant. Potash mobilized is immediately available to the plants. stimulates flowering and fruiting. Improves soil properties and sustains soil fertility. Encourages plumpness and succulence of fruits and grains and increases brix level. Increases the yield from 10% - 20% Improves the quality of produce and thus fetches good price.

19. Phosphorous Solubilizing Bio Fertilizers Phosphobacteria is one of the bio fertilizer. Phosphorus is a major nutrient for plants inducing vigorous growth and also contributing to their disease resistance. Phosphorous helps in root formation and plant growth. The plants utilize only 10–15% of phosphate applied. The balance 85 – 90% remains in insoluble form in the soil. The bio promoters has highly efficient phosphate solubilizing bacteria (Bacillus megaterium) that grow and secrete organic acids, which dissolve this unavailable phosphate into soluble form and make it available to the plants. Thus, the residual phosphate fertilizers in the soil can be well utilized and external application can be optimized. Advantages: Biopromoter facilitates root formation and plant growth. It improves soil quality with subsequent uses. It should not be mixed with antibacterial agents and inorganic fertilizers.

20. P Solubilizing Bio Fertilizers

21. P Mobilizing Bio Fertilizers

22. Zinc Solubilizing Bio Fertilizers Zinc Mobilizer is a bio fertilizer made of pure cultures of naturally occurring soil-borne zinc solubilizing bacterium. Zinc solubilizing bacterium multiplies, secrete organic acids and helps to solubilize insoluble and chelated zinc fixed in the  soil. This mobilized zinc is easily available to the plants. Examples: Bacillus

23. Vesicular Arbuscular Endomycorrhiza (VAM) VAM plays a great role in inducing plant growth. VAM are symbiotic entophytic soil fungi, which colonize the roots of approximately 80% plants. VAM fungi produce hyphae, which are microscopic tubes that colonize crop roots and grow out into the soil further than root hairs. Nutrients are taken up by the hyphae and carried back to the plant. This results into very efficient mobilization and uptake of fertilizers and other nutrients by plants.  The VAM hyphae also helps is retaining moisture around the root zone of plants. VAM increase the resistance to root borne or soil borne pathogens and nematodes. enhanced colonization of introduced population of beneficial soil organisms like Azotobacter, azospirillum, ehizobium and phosphate solubilizing bacteria around mycorrhizal roots thereby exerting synergistic effects on plant growth. Suitable for:        Turmeric, Banana, Rubber, Coffee, Tea, Pepper, Cardamom, Cocoa, Fruit trees, Tree seedlings and species etc. 

24. Mycorrhiza • Mycorrhizae are a group of fungi that include a number of types based on the different structures formed inside or outside the root. These are specific fungi that match with a number of favorable parameters of the host plant on which it grows. • This includes soil type, the presence of particular chemicals in the soil types, and other conditions. • These fungi grow on the roots of these plants. In fact, seedlings that have mycorrhizal fungi growing on their roots survive better after transplantation and grow faster. The fungal symbiont gets shelter and food from the plant which, in turn, acquires an array of benefits such as better uptake of phosphorus, salinity and drought tolerance, maintenance of water balance, and overall increase in plant growth and development. • While selecting fungi, the right fungi have to be matched with the plant. There are specific fungi for vegetables, fodder crops, flowers, trees, etc. • Mycorrhizal fungi can increase the yield of a plot of land by 30%-40%. It can absorb phosphorus from the soil and pass it on to the plant. Mycorrhizal plants show higher tolerance to high soil temperatures, various soil- and root-borne pathogens, and heavy metal toxicity.

26. Legume-rhizobium relationship • Leguminous plants require high quantities of nitrogen compared to other plants. • Nitrogen is an inert gas and its uptake is possible only in fixed form, which is facilitated by the rhizobium bacteria present in the nodules of the root system. • The bacterium lives in the soil to form rootnodules (i.e. outgrowth on roots) in plants such as beans, gram, groundnut, and soybean.

27. Blue-green algae Blue-green algae are considered the simplest, living autotrophic plants, i.e. organisms capable of building up food materials from inorganic matter. They are microscopic. Blue-green algae are widely distributed in the aquatic environment. Some of them are responsible for water blooms in stagnant water. They adapt to extreme weather conditions and are found in snow and in hot springs, where the water is 85 °C. Certain blue-green algae live intimately with other organisms in a symbiotic relationship. Some are associated with the fungi in form of lichens. The ability of blue-green algae to photosynthesize food and fix atmospheric nitrogen accounts for their symbiotic associations and also for their presence in paddy fields. Blue-green algae are of immense economic value as they add organic matter to the soil and increase soil fertility. Barren alkaline lands in India have been reclaimed and made productive by inducing the proper growth of certain blue-green algae.

28. How are Bio Fertilizers applied to crops? Seed treatment:200 g of nitrogenous bio fertilizer and 200 g of Phosphotika are suspended in 300-400 ml of water and mixed thoroughly. Ten kg seeds are treated with this paste and dried in shade. The treated seeds have to be sown as soon as possible. Seedling root dip: For rice crop, a bed is made in the field and filled with water. Recommended bio fertilizers are mixed in this water and the roots of seedlings are dipped for 8-10 hrs. Soil treatment: 4 kg each of the recommended bio fertilizers are mixed in 200 kg of compost and kept overnight. This mixture is incorporated in the soil at the time of sowing or planting.

29. How could one get good response to bio fertilizer application? • Bio fertilizer product must contain good effective strain in appropriate population and should be free from contaminating microorganisms. • Select right combination of bio fertilizers and use before expiry date. • Use suggested method of application and apply at appropriate time as per the information provided on the label. • For seed treatment adequate adhesive should be used for better results. • For problematic soils use corrective methods like lime or gypsum pelleting of seeds or correction of soil pH by use of lime. • Ensure the supply of phosphorus and other nutrients.

30. What would be probable reasons for not getting response from the application of bio fertilizers? • On account of quality of product • Use of ineffective strain. • Insufficient population of microorganisms. • High level of contaminants. • On account of inadequate storage facilities • May have been exposed to high temperature. • May have been stored in hostile conditions. • On account of usage • Not used by recommended method in appropriate doses. • Poor quality adhesive. • Used with strong doses of plant protection chemicals. • On account of soil and environment • High soil temperature or low soil moisture. • Acidity or alkalinity in soil. • Poor availability of phosphorous and molybdenum. • Presence of high native population or presence of bacteriophages.

31. What precautions one should take for using bio fertilizers? • Bio fertilizer packets need to be stored in cool and dry place away from direct sunlight and heat. • Right combinations of bio fertilizers have to be used. • As Rhizobium is crop specific, one should use for the specified crop only. • Other chemicals should not be mixed with the bio fertilizers. • While purchasing one should ensure that each packet is provided with necessary information like name of the product, name of the crop for which intended, name and address of the manufacturer, date of manufacture, date of expiry, batch number and instructions for use. • The packet has to be used before its expiry, only for the specified crop and by the recommended method of application. • Bio fertilizers are live product and require care in the storage • Both nitrogenous and phosphatic bio fertilizers are to be used to get the best results. • It is important to use bio fertilizers along with chemical fertilizers and organic manures. • Bio fertilizers are not replacement of fertilizers but can supplement plant nutrient requirements.

32. Bio Fertilizers Production Process • Preparation of Media for Culture • Zinc Solubilizing • Phosphate Solubilizing • Potassium Solubilizing • Nitrogen Solubilizing • For Each Medium, if preparing 100 ml of media add agar of 2.5 mg.

33. Phosphate Solubilizing Medium

34. Jensen's Media (Nitrogen Fixing)

35. Zinc Solubilizing Medium

36. Potassium Solubilizing Medium

38. Mother Culture Bacterial growth from original culture i.e. from lyophilized mineral slant is sub cultured on nutrient medium in petri plates and slants. The purpose is multiplication of bacteria. It includes transfer of isolated pure colonies from petri plates to test tubes, slants and further to broth (250-500 ml). Maintenance of culture by mineral oil. Lyophilisation and production of tube slant culture as per production requirements.

39. Preparation Of Media • All the media components are weighed accurately according to the values mentioned in the tables for 1 litre. Put Components accordingly in the flask. • In the media flask DM water is taken and all the components are dissolved in it. pH is adjusted. • For solid medium Agar is added and for liquid medium agar is not added. • Then it is kept for sterilization in the autoclave at 121 oC temperature and 1.5 kg per square cms pressure for 20 minutes • Media is cooled at room temperature.

40. Seed Culture Seed Culture is propagation of bacteria in broth (liquid media) in flasks of capacity 250 ml or 500 ml for inoculation. First Bacterial Suspension is prepared and loop full suspension is transferred to the broth in the flask under aseptic conditions. The flask is kept on shaker for shaking for 72 Hrs. It will help for rapid multiplication and population in the flask increases up to 108 cells per ml. Similarly bottle broth inoculation is done. This culture is used in the fermenter inoculation.

41. Bio Reactor Fermentation Final Culture is prepared in this step. Bio Reactor is operated with 2/3rd its capacity with proper aeration and agitation. Sterile medium in this vessel is inoculated with seed culture. It is mixed with agitator. Filtered air is allowed through filter for aeration. Fermentation is done for 72 hours. until population reaches up to 10 to the power of 8 per lt. Now this broth reaches for blending purpose. During this process micro organisms secretes some acids in the solution due to which pH decreases very much. It is maintained in the range of 6.5-7.5 by addition of 40% NaOH.

42. PRODUCT DETAILS Specification : Microbial Count : 1 X 108 cell/ml Properties: Color: Creamy Odor: Curdy Shelf Life: 1 year at 20 to 25 C Strain Used: Bacillus firmus Uses: Phosphate solubilizing bio fertilizers are recommend for all types of soil and crops. Therefore phosphate solubilizing bio fertilizers are having wide range of utility. Methods Of Application: For 1 acre, use 200 mL Biola mixed with 200 litres of water . This can be directly sprayed on the soil or broadcast with farm yard manure. Seed Application: Mix 15 ml Biola with 1 litre water and dip the seed for 30 minutes. Dry in Shade and use for sowing.

43. Advantages Of Biola: • Biola is eco friendly bio inoculant. • Enhance plant growth by release of vitamins, auxins and hormones. • 10-30% crop yield increase with use. • Improve physical, chemical and biological properties of soil. • Enhance seed germination. • Enhance plant flowering and maturity. • Increases phosphate uptake efficiency. • No harmful effect on soil fertility and plant growth. • Plant growth promotions.

44. Development and Marketing Of Bio Fertilizers OVERVIEW: For the past four decades or so farmers have been using chemical fertilizers as an important farm input apart from seeds, farm implements, etc. For the farmer, a fertilizer is a substance, which provides vital nutrients for plant growth. The primary nutrients required for plant growth are nitrogen, phosphorus and potassium. Chemical fertilizers are chemical compounds, which provide these primary nutrients and therefore have a fixed formula. Chemical fertilizers are applied to soil in order to enrich it with required nutrients. Results of chemical fertilizer usage are easily and immediately visible in terms of increased yield per hectare. However, in the long run, they show diminishing returns.

45. Problems Regarding The Marketing Of Bio Fertilizers: • Lack of awareness of: • Farmers • Dealers • Officers • Extension Workers • Difficulty in understanding and usage of Bio Fertilizers by the Farmers • NON AVAILABILITY OF BIO FERTILIZERS: • Bio-fertilizers are distributed through the Gram Panchayats. However, 3 out of 4 farmers who are currently using bio-fertilizers say that they are not available through the Gram Panchayat. The government sells bio-fertilizers at subsidized rates to farmers through its own distribution mechanism consisting of Block Development Officers, TalukaKrishiAdhikaris, Gram Sevaks and other types of extension workers who promote the Integrated Nutrient Management Programme.  Bio-fertilizers are also available through the dealer network, but very few dealers stock bio-fertilizers.

46. Results are not immediately seen • Results of bio-fertilizer usage are not immediately apparent and not easily attributable.  3 out of 4 farmers, who were aware of bio-fertilizers, feel that results are slow. Farmers classify results of bio-fertilizer usage under 3 heads: increase in soil fertility, increase in yield, and improvement in quality of produce. Without an exception, all farmers feel that results of bio-fertilizer usage are slow. • Role Of the Government • Without exception, all farmers said that they have not got any information about bio-fertilizers from Government agencies, like Gram Sevaks and Panchayat Samitis.

47. Examples of New Commercial Microbial Inoculants Commercial microbial inoculants of other beneficial microorganisms have begun to appear on the market on a small scale. These include "Azogreen", a French-approved Azospirillum inoculant. COST OF DEVELOPMENT AND MARKETING The cost of developing a new product by the agrochemical industry has been estimated at over $80 million US and rising . The development of resistance to pesticides may shorten the commercial life of these products and thus their potential return. The development of bacterial inoculants is claimed to be cheaper than that of agrochemicals.

48. The following are some factors that reduce the costs of development of bacterial inoculants which makes them attractive to the agrochemical industry: (i) Reduced registration costs compared to those of chemical-product test programs t hat are well- established and costly. (ii) Reduced registration time decreases the time span from first screening to market, thus increasing revenues (iii) The possibility of developing bacterial products for small markets. Since the cost involved in bringing a new chemical to the marketplace is so large, the product must be targeted to a market large enough to have a good return on investment. This limits the choice of crops to the major crops only. (iv) Although fermentation is costlier than chemical production, the fermentation plant is more versatile.

49. Other motivational steps for the agrochemical industry to develop bacterial inoculants might be: • It is less likely that pathogens will develop resistance as fast as they do to chemical products. • Some bacterial inoculants, especially those that use an organism employing a single mechanism against the pathogen, can also develop resistance. • They are "environment friendly". The "natural“ tag of bacterial inoculants (especially those that are non engineered and indigenous) make them more acceptable in the public eye, and especially t o the "Green movement" pressure groups, than chemicals.

50. Market requirement First, all the considerations mentioned above (efficient strains, optimized formulations, cost-effective production, and good and practical inoculation techniques) are not sufficient to launch a new product on the market nor guarantee its success. The following practical variables should be considered: (i) The product must be efficient and reliable in large-scale field trials and especially under "real life" conditions. (iii) Obviously, patents on industrial processes and registration of biological products must be secured (iv) For every potential customer country, a market survey must be done which examines customer demand, market size, and expected selling price.